Regulation of lipolysis and lipogenesis in adipose tissue in the state of insulin resistance

Inzulinska rezistenca (IR) je stanje zmanjšanega vstopa glukoze v tarčna tkiva pri odzivu na inzulin. Zaradi tega je za vzdrževanje normalne plazemske koncentracije glukoze potrebna zvečana koncentracija inzulina. IR je povezana z mnogimi bolezenskimi stanji, kot so metabolični sindrom, sindrom policističnih jajčnikov (PCOS), diabetes tipa 2 in lipodistrofije. IR in hiperinzulinemija vodita do nevarnih srčno-žilnih zapletov. Osnovni dejavnik v razvoju IR je centralna debelost, kopičenje visceralnega maščobnega tkiva. Eden glavnih vzrokov za razvoj IR je povečano sproščanje prostih maščobnih kislin iz maščobnega tkiva, poleg tega pa so vpleteni še drugi mehanizmi, npr. spremenjeno izločanje adipokinov. Sproščanje prostih maščobnih kislin uravnavata dva osnovna procesa v maščobnem tkivu: lipogeneza in lipoliza. V pričujoči doktorski disertaciji smo kot modelno skupino za IR obravnavali bolnice s PCOS in proučili vlogo genov za nekatere pomembne encime lipogeneze oziroma sinteze trigliceridov: lipoproteinsko lipazo (LPL), lipin 1 (LPIN1), ter gene, povezane z lipolizo: gen za hormonsko-senzitivno lipazo (LIPE) in gen za encim, ki reaktivira kortizol – 11β-hidroksisteroidno dehidrogenazo tipa 1 (HSD11B1), saj glukokortikoidi spodbujajo lipolizo in imajo še druge inzulinu nasprotne učinke. Poleg tega smo proučili tudi izražanje gena za jedrni receptor PPARγ (s peroksisomskim proliferatorjem aktiviran receptor γ), ki spodbuja adipogenezo in sintezo trigliceridov. Dodatno smo analizirali še dva gena, ki sta povezana z inzulinsko občutljivostjo: gen za adiponektin (zaščitni dejavnik iz adipocitov) in gen za glukozni prenašalec GLUT4. Zaradi posebne povezave centralne debelosti z IR in poročil o različni presnovni vlogi dveh osnovnih tipov maščobnega tkiva smo proučevali izražanje genov v visceralnem in subkutanem maščobnem tkivu. V raziskavo subkutanega maščobnega tkiva smo vključili 85 bolnic s PCOS po kriterijih National Institutes of Health in 44 zdravih žensk, na manjši skupini (30 bolnic in 25 zdravih žensk) pa smo proučevali visceralno maščobno tkivo, pridobljeno z laparoskopijo. Vsem preiskovankam smo izmerili antropometrične kazalce in laboratorijske presnovne kazalce. 34 bolnic se je 6 mesecev zdravilo z učinkovinami za izboljšanje inzulinske občutljivosti, bodisi z agonistom PPARγ rosiglitazonom bodisi z metforminom. Pri bolnicah in zdravih ženskah smo proučili bazalno raven izražanja genov v povezavi s presnovnim stanjem ter pri omenjeni skupini bolnic še spremembe izražanja genov tekom zdravljenja. Raven mRNA omenjenih genov smo merili z metodo PCR v realnem času in za normalizacijo uporabili dva hišna gena. Dodatno smo pri 223 bolnicah s PCOS in 149 zdravih ženskah analizirali genomsko DNA na polimorfizme v genih PPARG, LPIN1 in HSD11B1. Ugotovili smo, da je pri bolnicah s PCOS glede na zdrave ženske zavrto izražanje gena LPIN1 v visceralnem in subkutanem maščobnem tkivu, kar zniža lipogenezo v teh tkivih in verjetno preusmeri lipide v druga tkiva ter prispeva k IR. Izražanje HSD11B1 je pri bolnicah s PCOS v obeh tipih maščobnega tkiva zvišano. V visceralnem maščobnem tkivu povečane količine glukokortikoidov inducirajo gene LPL, PPARG in LIPE, kar spodbuja razvoj centralne debelosti in sproščanje prostih maščobnih kislin v krvni obtok. V subkutanem maščevju glukokortikoidi zavirajo izražanje adiponektina. Tako glukokortikoidi z delovanjem v obeh tkivih poslabšajo IR. V subkutanem maščevju je pri PCOS zavrto izražanje genov za kopičenje lipidov: LPL in PPARG, kar dodatno preusmerja maščobe v bolj nevaren visceralni maščobni depo. Poleg tega je v subkutanem maščobnem tkivu zavrto tudi izražanje lipolitičnega gena LIPE, kar bi lahko predstavljalo prilagoditveni mehanizem organizma, ki ščiti pred pretiranim sproščanjem prostih maščobnih kislin. Agonist PPARγ poveča izražanje LPL in zniža izražanje HSD11B1 v subkutanem maščobnem tkivu. Tako se pri zdravljenju subkutano poveča lipogeneza in zmanjša negativen vpliv glukokortikoidov, kar izboljša inzulinsko občutljivost. Mehanizmi uravnavanja lipogeneze in lipolize se torej med obema tipoma maščobnega tkiva kvalitativno razlikujejo. Med subkutanim in visceralnim maščobnim tkivom korelirata le ekspresiji genov LPIN1 in HSD11B1, za proučevanje drugih genov je potrebna analiza vsakega tkiva posebej. Mutirani genotipi polimorfizmov v genu PPARG, Pro12Ala in 1431C>T so ugoden dejavnik za manjšo stopnjo debelosti. Mutirani genotipi polimorfizmov -681C>G in Pro12Ala v genu PPARG izboljšajo učinek zdravljenja z rosiglitazonom. Polimorfizma SNP1 in SNP17 v genu LPIN1 imata nasproten vpliv na kazalce IR. Heterozigoti za polimorfizem IVS3+53_54insA v genu HSD11B1 imajo najnižje izražanje HSD11B1 v subkutanem maščobnem tkivu in najugodnejše presnovno stanje med vsemi genotipi. Zaključimo lahko, da gena HSD11B1 in PPARG, ki modulirata delovanje transkripcijskih faktorjev (glukokortikoidov in receptorjev PPARγ), pomembno uravnavata lipogenezo in lipolizo po različnih mehanizmih v visceralnem in subkutanem maščevju ter tako vplivata na IR.Insulin resistance (IR) is a state in which target cells fail to uptake glucose at ordinary levels of plasma insulin. Hence, higher than normal insulin concentrations are needed to maintain euglycemia. IR is associated with various pathological conditions, e. g. metabolic syndrome, polycystic ovary syndrome (PCOS), type 2 diabetes and lipodystrophies. IR and hyperinsulinemia finally lead to serious cardiovascular complications. Central obesity with accumulation of visceral adipose tissue has been recognized as the most common cause of IR. One of the main mechanisms is increased free fatty-acid release from adipose tissues. Additionally, altered adipokine secretion is involved. Free fatty acid release is regulated by two fundamental processes: lipogenesis and lipolysis. In the present doctoral dissertation, the PCOS patients as a model disease group were used. The genes for some important enzymes involved in lipogenesis - triglyceride synthesis were studied: lipoprotein lipase (LPL) and lipin 1 (LPIN1) along with the genes associated with lipolysis: the gene for hormone-sensitive lipase (LIPE) and the gene for glucocorticoid reactivating enzyme 11β-hydroksisteroid dehydrogenase type 1 (HSD11B1), as glucocorticoids augment lipolysis and act as insulin antagonists. Further, the expression of peroxisome-proliferator acivated receptor γ gene (PPARG) was studied due to its role in adipogenesis and triglyceride synthesis. Another two genes involved in providing insulin sensitivity were analyzed: adiponectin gene (a protective factor from adipocytes) and glucose transporter 4 (GLUT4) gene. As central obesity is particularly associated with IR, the expression of genes in visceral and subcutaneous adipose tissues was explored. We enrolled 85 PCOS patients according to the National Institutes of Health criteria and 44 healthy women for subcutaneous adipose tissue biopsy and 30 patients and 44 controls for visceral adipose tissue biopsy. In all study subjects, anthropometric and metabolic markers were measured. Thirty-four PCOS patients underwent 6-month insulin sensitizing therapy with either PPARγ agonist rosiglitazone or metformin. In patients and controls the basal levels of gene expression in association with metabolic state and, in the aforementioned patients, the changes after the therapy were measured. Gene expression was measured by real time PCR and two housekeeping genes were usedfor normalization. In PCOS patients, decreased LPIN1 expression was found as compared to healthy controls, in visceral and subcutaneous adipose tissue. This could decrease lipogenesis in adipose tissues shifting lipids to liver and muscle and contributing to IR. HSD11B1 expression was increased in both types of adipose tissue. In visceral fat, increased amounts of glucocorticoids induce LPL, PPARG and LIPE genes, which stimulate the development of central obesity and free fatty acid release into the circulation. In subcutaneous fat, adiponectin expression is attenuated by glucocorticoids. Thus, IR is exaggerated by glucocorticoids in both types of adipose tissue. There is inhibited expression of lipid accumulation genes in subcutaneous adipose tissue in PCOS patients: LPL and PPARG, which additionally shifts lipids to less safe visceral depot. Additionally, the expression of lipolysis LIPE gene is diminished in subcutaneous fat, which could represent an adaptive mechanism in obesity to repress free fatty acid release. PPARγ agonist augments LPL expression and reduces HSD11B1 expression in subcutaneous depot. In this way lipogenesis is increased in subcutaneous fat and the negative impact of glucocorticoids is reversed. The mechanisms of lipogenesis and lipolysis differ qualitatively between the two types of adipose tissue. Only LPIN1 and HSD11B1 show correlation between expression in visceral and subcutaneous adipose tissue, all other genes need to be analyzed in particular type of fat.The genotype analysis showed that mutated -681C>G and Pro12Ala genotypes of PPARG gene favor a leaner phenotype. Mutated genotypes of -681C>G and Pro12Ala polimorphisms augment the beneficial effects of rosiglitazone treatment. SNP1 and SNP17 polimorphisms in LPIN1 have mutually opposite effects on IR markers. Heterozygotes of IVS+53_54insA polymorphism in HSD11B1 gene have the lowest HSD11B1 expression in subcutaneous adipose tissue and the most favorable metabolic profile. We can conclude that HSD11B1 and PPARG genes which modulate the action of glucocorticoids and PPARγ transcription factors play a crucial role in modifying IR by regulating lipogenesis and lipolysis. The mechanisms of this regulation in visceral and subcutaneous adipose tissue are considerably different